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O
O
Gly67
N
N
Tyr66
H
cy clization
NH
NH
O
OH
OH
OH
Ser65
OH
OH
dehydration
H 2 O
O
O
H 2 O 2
O 2
GFPn
N
N
N
N
oxidation
OH
OH
OH
OH
O
GFPa
N
N
O
OH
Fig. 2 Proposed mechanisms of biosynthesis of the GFP chromophore. In the last step of the
reaction, the two relevant protonation states are shown
The mature chromophore consists of two rings, the phenol ring, coming from the
side chain of Tyr66, and the five-member heterocyclic ring (imidazolinone), resulting
from the cyclization of the backbone. The imidazolinone core is a common feature of
all known FP chromophores. The alternating single and double bonds in the bridge
region extend the electron delocalization from the phenolate to the carbonyl of the
imidazolinone. Efficient visible-light absorption is ultimately determined by this
p
-conjugated system, i.e., the single-double bond alternation with connected atomic
p-orbitals.
2.2 Absorption, Excitation, and Emission
The room temperature absorption, excitation, and emission spectra of av GFP are
shown in Fig. 3 . Apart from the 278-nm band, which is common to proteins
containing aromatic amino acids, av GFP exhibits a major absorption band at
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